As advances continue to be made in the molecular biology of inherited or acquired diseases, modulation or modification of the genetic program of living cells is looked upon with growing interest as a new therapeutic approach. Two different strategies have emerged: Gene therapy and oligonucleotide-based therapeutics. To be successful these two approaches must be mediated by an efficient "in vivo" transfer of the nucleic acid material to the target cells and there is a need to provide an efficient and safe delivery system of nucleic materials.
Gene therapy involves the transfer of normal, functional genetic material into cells to correct an abnormality due to a defective or deficient gene product. Typically, the genetic material to be transferred should at least contain the gene to be transferred together with a promoter to control the expression of the new gene. In general, two gene transfer strategies are considered: Viral (retrovirus or adenovirus) vectors and synthetic gene-transfer vectors.
Viral agents have been demonstrated to be highly efficient vectors for the transfection of somatic cells. Retroviruses in particular have received a great deal of attention because they not only enter cells efficiently, but also provide a mechanism for stable integration into the host genome through the provirus. However, clinical use of retroviral vector is hampered by safety issues. A first concern is the possibility of generating an infectious wild type virus following a recombination event. A second concern is the consequences of the random integration of the viral sequence into the genome of the target cell which may lead to tumorigenic event. In addition, as retroviruses would only complete their life cycle in dividing cells, a retroviral vector would be inefficient in targeting cells which are not dividing. DNA viruses such as adenoviruses are potential gene carriers but this strategy is limited in the size of the foreign DNA adenoviruses can carry and because of the restricted host range. However, the advantage of adenoviruses over retroviral vectors is their ability to infect post-mitotic cells.
Synthetic gene-transfer vectors have been subject to intense investigation since this strategy appears to be clinically safe. Potential methods of gene delivery that could be employed include DNA/protein complexes (1) or liposomes (2-7). The genetic material to be delivered to target cells by these methods are plasmids. Plasmids are recombinant DNA which are circular forms of double stranded DNA. They are constructed so that they have, at the minimum, a promoter and the gene coding for the protein of interest. Plasmids can be expressed in the nucleus of the transfected cells in a transient manner. In rare events, the plasmids may be integrated or partly integrated in the cell host genome and might therefore be stably expressed. Episomal plasmid vectors are plasmids able to replicate in the nucleus of the transfected cells and may therefore be expressed in a total growing cell population. Plasmids may have a promising potential considering the fact that they may be applied in combination with a synthetic vector as carrier and that gene therapy by this means may be safe, durable, and used as drug-like therapy.
Plasmid preparation is simple, quick, safe, and inexpensive representing important advantages over retroviral vector strategy. The successful use of this genetic tool for "in vivo" approaches to gene therapy will rely on the development of an efficient cell delivery system.
Liposomes have been shown to be efficient vehicles for many in vitro and in vivo applications. Liposome encapsulated DNA have been used in vitro (3,5) and in vivo (2,4,6,7) for the expression of a given gene through the use of plasmid vectors. The term "liposomes" describes a closed structure composed of lipid bilayers surrounding an internal aqueous space. Liposomes may be used to package DNA for delivery to cells, even in the case of plasmids of large size which could potentially be maintained in a soluble form that would allow direct application to in vivo systems by a simple intravenous injection. Formation of complexes of DNA with cationic liposomes has recently been the focus of research of many laboratories. In particular, lipofectin (Gibco BRL, Gaithersburg, Md.) has been successfully used for the transfection of various cell lines in vitro (3) and for systemic gene expression after intravenous delivery into adult mice (6).
Lipofectin is formed with the cationic lipid DOTMA, N1-(2,3-dioleyloxy) propyl!-N,N,N-trimethyl-ammonium chloride, and DOPE, dioleylphosphatidyl ethanolamine at a 1:1 molar ratio. The liposomes prepared with this formulation spontaneously interact with DNA through the electrostatic interaction of the negative charges of the nucleic acids and the positive charges at the surface of the cationic liposomes. This DNA/liposomal complex fuses with tissue culture cells and facilitates the delivery of functional DNA into the cells (3). New cationic liposomes have been developed: Lipofectamine (Gibco BRL), composed of DOSPA, 2,3-dioleyloxy-N2(sperminecarboxamido)ethyl!-N,N-dimethyl-1-propanaminium trifluoracetate and DOPE at a 1:1 molar ratio. Lipofectace (GIBCO BRL) composed of DDAB, dimethyidioctadecylammonium chloride and DOPE at a 1:1 molar ratio. DOTAP (Boehringer Mannheim, Ind.) 1-2-dioleoyloxy-3 (trimethyl ammonia) propane.
Behr et al. (9,10) have recently reported the use of a lipopolyamine (DOGS, Spermine-5-carboxy-glycinediotadecylamide) to transfer DNA to cultured cells. Lipopolyamines are synthesized from a natural polyamine spermine chemically linked to a lipid. For example, DOGS is made from spermine and dioctadecylamidoglycine (9). DOGS spontaneously condence DNA on a cationic lipid layer and result in the formation of nucleolipidic particles. This lipospermine-coated DNA shows high transfection efficiency (10).
It is an object of the present invention to provide an efficient and safe delivery system for nucleic acid materials.